Combination Treatment of Cardiovascular Disease

ABSTRACT

Disclosed are methods, compositions of matter and cells for treatment of cardiovascular disease through concurrent inhibition of oxidative stress while administration of a cell therapy. The invention also concerts the modulation of oxidative stress for preferential induction of differentiation while concurrently inhibiting inflammatory processes that decrease efficacy of cellular therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a continuation ofNon-Provisional application Ser. No. 12/108,130, filed Apr. 23, 2008 andentitled “Combination Treatment of Cardiovascular Disease” which claimspriority to Provisional Application Ser. No. 60/913,531, filed Apr. 23,2007 and entitled “Combination Treatment of Cardiovascular Disease”,both of which are expressly incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The invention relates to treatment of cardiovascular disease.Particularly, the invention relates to methods of treatment comprisingof, inter alia, combination treatment of cells and antioxidants. Moreparticularly, the invention relates to utilization of antioxidants toenhance efficacy of cells with ability to ameliorate or significantlyreduce cardiovascular disease.

BACKGROUND

Acute myocardial infarction (heart attack) is a major cause of morbidityand mortality with a reported annual incidence of 1.1 million cases inthe United States alone. It is despite the increasing use of cholesterollowering agents and attentiveness to co-morbid illnesses. Subsequent toinfarction, a variety of inflammatory and other changes are known tooccur which despite proper reperfusion by thrombolytics and/or stenting,contribute to cardiac remodeling and eventual heart failure. Numerousother cardiac conditions are widely prevalent, particularly myocardialischemia which is associated with atherosclerosis of arteries feedingmyocardial tissue. Congenital and acquired cardiac abnormalities arenumerous and range from valvular defects to hypertrophy to septaldefects.

Cellular therapy of cardiovascular diseases has achieved some degree ofsuccess. For example, administration of autologous bone marrow stemcells has been demonstrated to benefit patients with end-stage chronicischemic cardiomyopathy (1, 2). Additionally, administration of similarstem cell populations subsequent to the stunning phase of acutemyocardial infarction has been demonstrated to induce an increase inleft ventricular ejection fraction as compared to control patients (3).The methods by which stem cells induce therapeutic effect incardiovascular diseases include induction of angiogenesis (4),inhibition of ventricular remodeling (5), and transdifferentiation intocardiomyocytes (6). Additionally, besides stem cells, skeletal musclecells have also been used for treatment of cardiovascular diseases (7).

Numerous patents have been issued on utilizing stem cells for treatmentof cardiovascular disease. For example, U.S. Pat. No. 7,166,280 entitled“Combination growth factor therapy and cell therapy for treatment ofacute and chronic heart disease” teaches the combination of growthfactor administration together with stem cell administration. Some ofthe growth factors mentioned in the patent have already been used fortreatment of heart disease such as FGF and VEGF members. Additionally,it is important to note that others have already demonstrated synergybetween administration of these types of growth factors together withstem cells. U.S. Pat. No. 6,387,369 entitled “Cardiac muscleregeneration using mesenchymal stem cells” discloses the use ofmesenchymal stem cells for cardiac repair, specifically after myocardialinfarction.

To date, no combination therapy has been reported that concurrentlyinhibits oxidative stress and administers stem cells. Although reportsexist of utilizing nutritional intervention together with stem celltherapy (8), these do not induce substantive antioxidant effect. Giventhat stem cells are known to be sensitive to oxidative stress, thecurrent invention provides, inter alia, a method of increasing efficacyof stem cell therapy through concurrent administration of antioxidantswith said stem cell therapy.

SUMMARY

Embodiments herein relate to methods of treating cardiovascular diseasecomprising: a) identifying a subject suffering from cardiovasculardisease; b) administering one or more cell populations capable ofameliorating cardiovascular disease to said subject in a sufficientamount such that said cardiovascular disease is ameliorated; and c)administering an antioxidant to subject in sufficient amount to enhancethe amerlioration of cardiovascular disease by said one or more cellpopulations.

The term cardiovascular disease, as used herein can non-exclusively beselected from a group consisting of: cardiomyopathy, post myocardialinfarction scarring, myocardial ischemia, coronary artery disease,peripheral vascular disease, CNS vascular disease, congestive heartfailure, ventricular septal defect, a valvular defect, atrial septaldefect, a congenital heart defect, ventricular aneurysm, a conditionrequiring ventricular reconstruction, restenosis, cardiac hypertrophy,and heart failure.

Preferably, the one or more cell populations capable of amelioratingcardiovascular disease are selected from the group consisting of:differentiated cells, progenitor cells, and stem cells. Advantageously,differentiated cells can be selected from the group consisting of:myocytes, cardiomyocytes, and striated muscle cells. In furtherembodiments, progenitor cells can be selected from the group consistingof: endothelial progenitor cells, cardiovascular progenitor cells, andhematopoietic progenitor cells. In other embodiments, stem cells can beselected from the group consisting of: embryonic stem cells, cord bloodstem cells, placental stem cells, bone marrow stem cells, amniotic fluidstem cells, amniotic membrane stem cells, menstrual blood derived stemcells, endometrial regenerative cells, neuronal stem cells, circulatingperipheral blood stem cells, mesenchymal stem cells, germinal stemcells, adipose tissue derived stem cells, exfoliated teeth derived stemcells, hair follicle stem cells, dermal stem cells, parthenogenicallyderived stem cells, reprogrammed stem cells and side population stemcells.

The one or more cell populations provided herein can include bothmesenchymal stem cells and CD34 cells.

Preferred antioxidants used herein can be selected from the groupconsisting of: ascorbic acid and derivatives thereof, alpha tocopheroland derivatives thereof, rutin, quercetin, ascorbic acid, allopurinol,hesperedin, lycopene, resveratrol, tetrahydrocurcumin, rosmarinic acid,Ellagic acid, chlorogenic acid, oleuropein, alpha-lipoic acid,glutathione, polyphenols, pycnogenol, retinoic acid, ACE InhibitoryDipeptide Met-Tyr, recombinant superoxide dismutase, xenogenicsuperoxide dismutase, and superoxide dismutase, for example.

In preferred embodiments, the antioxidant can be administered to thesubject prior to, concurrently with, or subsequent to the administrationof said one or more cell populations and at a concentration sufficientto reduce oxidative stress from inhibiting the ameliorating effects ofsaid one or more cell populations on said cardiovascular disease.

Further methods can include measuring the oxidative stress in thesubject prior to the administration of said one or more cellpopulations, and wherein the antioxidant is administered at aconcentration and frequency based upon said measurement of oxidativestress.

An additional method of treating cardiovascular disease can include: a)identifying a subject suffering from cardiovascular disease; b)administering a mesenchymal stem cell population to said subject; b)administering a CD34 positive stem cell population to said subject; andc) administering ascorbic acid intravenously to said subject in acombined amount sufficient to ameliorate said cardiovascular disease.

The mesenchymal stem cell population preferably expresses one or moremarkers selected from the group consisting of: STRO-1, CD105, CD54,CD106, HLA-I markers, vimentin, ASMA, collagen-1, fibronectin, LFA-3,ICAM-1, PECAM-1, P-selectin, L-selectin, CD49b/CD29, CD49c/CD29,CD49d/CD29, CD61, CD18, CD29, thrombomodulin, telomerase, CD10, CD13,STRO-2, VCAM-1, CD146, and THY-1. According to more specificembodiments, the mesenchymal stem cell population does not expresssubstantial levels of the markers selected from the group consisting of:HLA-DR, CD117, and CD45.

The mesenchymal stem cell population can be derived from sourcesselected from the group consisting of: bone marrow, adipose tissue,umbilical cord blood, placental tissue, peripheral blood mononuclearcells, differentiated embryonic stem cells, and differentiatedprogenitor cells, for example.

According to other embodiments, the CD34 positive stem cell populationpossesses angiogenic activity. Additionally, the CD34 positive stem cellpopulation can possess hematopoietic activity. CD34 positive stem cellpopulation can be derived from multiple sources, non-exclusivelyincluding: mobilized peripheral blood, peripheral blood, cord blood,bone marrow, and embryonic stem cells. The CD34 positive stem cellpopulation can be expanded in vitro prior to adminstration to subject,according to advantageous methods.

Preferably, the ascorbic acid is administered to the subject at afrequency and concentration sufficient to enhance the amelioratingeffects of said mesenchymal stem cell population and said CD34 positivestem cell population. For example, the ascorbic acid can be administeredto said subject intravenously at 15-700 grams per week. According topreferred embodiments, the ascorbic acid is administered together withlipoic acid and/or a water soluble salt of lipoic acid. A preferredadministration of ascorbic acid is 100-1000 milligrams per day togetherwith lipoic acid and/or a water soluble salt of lipoic acid.Alternatively, the ascorbic acid can be administered to said subject at300-600 milligrams per day together with lipoic acid and/or a watersoluble salt of lipoic acid. In advantageous embodiments, the ascorbicacid is administered to the subject together with lipoic acid and/or awater soluble salt of lipoic acid at a respective ratio of 1:1 to3500:1. In still further embodiments, the ascorbic acid is administeredto the subject together with lipoic acid and/or a water soluble salt oflipoic acid at a respective ratio of 10:1 to 100:1. The methods hereincan further include administering one or more growth factors to saidsubject.

DETAILED DESCRIPTION

The invention teaches methods of treating cardiovascular disease throughadministration of cells with cardio-reparative potential in anenvironment that has been modified through administration of one or moreantioxidants. It is known that numerous cardiovascular conditions areassociated with increased levels of oxidative stress and inflammatorychanges. For example, circulating levels of C-reactive protein (CRP), amarker of inflammation are associated with extent of atherosclerosis(9). Elevated levels of CRP are also predictive of coronary heartdisease and found increased in valvular heart disease (10, 11). It isbelieved that causes of inflammation are associated with increasedoxidative stress, and in some cases said oxidative stress is actuallycausative of inflammation. For example, an inverse correlation has beendemonstrated between plasma ascorbic acid and CRP levels in patientswith peripheral artery disease (12). Administration of ascorbic acid andvarious other antioxidants has been demonstrated to decrease CRP levelsin patients with a variety of inflammatory associated conditions (13,14).

Administration of cellular therapy for treatment of cardiovasculardisease is based on the notion of inducing angiogenesis, and/or inducingdifferentiation into functional tissue, and/or providing trophic supportfor endogenous cells to replace damaged tissue. The introduction ofcells to a patient with cardiovascular disease implies cells areimplanted in an environment associated with inflammation. For example,subsequent to myocardial infarction, oxidative stress from the directischemia reperfusion injury, as well as subsequent cellularinfiltration, is associated with increased scar tissue formation andsubsequent pathological remodeling (15). Various sources of oxidativestress have been implicated including mitochondria, xanthine oxidase andthe non-phagocytic NADPH oxidases (16). There is evidence to suggestthat administration of antioxidant agents inhibit pathologicalremodeling (17-21). There is also evidence to suggest thatadministration of various types of cells into injured myocardium, orsystemically may inhibit pathological remodeling (22, 23). However, todate, there has been no concurrent inhibition of inflammatory responsestogether with cellular therapy. The importance of the combination isthat inflammatory agents are often inhibitory to stem cell activity. Forexample, it is known that inflammatory agents such as TNF-alpha inhibitability of stem cells to self renew (24, 25). Additionally, stem cellsare known to be particularly sensitive to oxidative stress (26, 27). Theparticular sensitivity of stem cells to oxidative stress may explaintheir increased viability and function under conditions of hypoxia(28-32). Accordingly, in an embodiment of the invention, cells withpotential to repair cardiovascular tissue is used in conjunctionantioxidant administration in order to induce repair of cardiovasculardisease.

In a specific embodiment, patients suffering a myocardial infarction arerevascularized using procedures known in the art. Said proceduresinclude administration of thrombolytics such as tissue plasminogenactivator (TPA) and/or introduction of a single or plurality of stentsin order to allow perfusion of the infarct related artery. Subsequent torevascularization, said patients are treated with cells capable ofcausing cardiac repair. Said cells may be, in one embodiment,mesenchymal stem cells. It is known in the art that mesenchymal stemcells induce both anti-inflammatory effects, as well as ability toprovide trophic factors that accelerate muscle repair. In conjunctionwith mesenchymal stem cells, patients are treated with expanded CD34cells. Without being bound to theory, the combination of mesenchymalstem cells and CD34 cells are used to concurrently induce angiogenesis,as well as provide healing and reparative growth factors. In conjunctionwith cell administration, patients are treated with antioxidants. In oneembodiment intravenous ascorbic acid is administered concurrently withcell therapy. In specific embodiments it is necessary to provideintravenous ascorbic acid in order to attain a higher concentration ofascorbic acid in systemic circulation than can be achieved through othermeans of administration such as oral. It was recently published thatonly intravenous administration of ascorbic acid, but not oral, canattain certain levels of plasma ascorbic acid necessary to inducepharmacological concentrations of ascorbate in the plasma (33). In otherstudies it was demonstrated that intravenous administration of ascorbicacid was able to achieve a 140-fold higher dose than those from maximumoral doses (34). The need in some cases to use intravenousadministration is due to the tight control of plasma ascorbic acidduring oral administration. Depending on clinical outcome, additionalcells and/or antioxidants may be provided. In one embodiment,mesenchymal stem cells are provided in absence of cord CD34 cells withthe purpose that mesenchymal stem cells will inhibit inflammation andfunction with enhanced benefit in the presence of one or moreantioxidants. In one embodiment, CD34 cells are provided in absence ofmesenchymal stem cells with the purpose that CD34 cells will induceangiogenesis with enhanced benefit in the presence of one or moreantioxidants. In some embodiments the therapy is performed incombination with a growth factor or a plurality of growth factors. Thisincludes, without limitation, angiogenic factors and other moleculescompetent to induce angiogenesis, including acidic and basic fibroblastgrowth factors, vascular endothelial growth factor, hif-1, epidermalgrowth factor, transforming growth factor .alpha. and .beta.,platelet-derived endothelial growth factor, platelet-derived growthfactor, hepatocyte growth factor and insulin like growth factor; growthfactors; BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8,BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, and BMP-16.Dependent on embodiment, cells, and/or growth factors, and/orantioxidants may be provided intravascularly, intravenously,intraarterially, intraperitoneally, via intraventricular infusion, viainfusion catheter, via balloon catheter, via bolus injection, or viadirect application to tissue.

In another embodiment, the invention provides the use of combinedadministration of stem cells with antioxidants for the treatment ofischemic heart disease. It is known that in ischemic heart disease anincreased level of oxidative stress is present (35). Accordinglypatients with ischemic heart disease are treated by combined use ofcells such as combination of CD34 and mesenchymal stem cells asdescribed above, along with the administration of one or moreantioxidants. Various other cardiovascular conditions are amenable totreatment with the current invention; these include cardiomyopathy, postperipheral vascular disease, CNS vascular disease, congestive heartfailure, ventricular septal defect, valvular defects, atrial septaldefect, congenital heart defect, ventricular aneurysm, a conditionrequiring ventricular reconstruction, restenosis, cardiac hypertrophy,and heart failure.

Example 1

50 patients with congestive heart failure (left ventricular ejectionfraction <35%) are entered into a clinical trial. 25 are treated withplacebo and stem cells, 25 receive stem cells and intravenous ascorbicacid (active treatment). Active treatment comprises of 25 grams ofintravenous ascorbic acid given in 250 ml of saline intravenously. Theascorbic acid is allowed to drip for 30 to 40 minutes into the patient.4 hours after, 5 million CD34 cells derived from cord blood are givenintravenously and 3 million mesenchymal stem cells are givenintravenously. Cells are given for 4 consecutive days. Followed up a 2day rest period. On day 7 cells are administered again, 5 million CD34and 3 million mesenchymal cells, along with the same concentration ofascorbic acid as given on day 1. After a period of 4 weeks animprovement is seen ejection fraction and clinical heart failure scorein both groups as compared to pre-treatment. Improvement issignificantly higher in the patients who have received ascorbic acidtogether with the stem cells.

Mesenchymal cells are prepared as described in as described in Meng etal. Endometrial regenerative cells: a novel stem cell population. JTransl Med. 2007 Nov. 15;5:57). CD34 cells are extracted and expanded asdescribed below.

Umbilical cord blood is purified according to routine methods((Rubinstein, et al. Processing and cryopreservation ofplacental/umbilical cord blood for unrelated bone marrow reconstitution.Proc Natl Acad Sci U S A 92:10119-10122). Briefly, a 16-gauge needlefrom a standard Baxter 450-ml blood donor set containing CPD Aanticoagulant (citrate/phosphate/dextrose/adenine) (Baxter Health Care,Deerfield, Ill.) is inserted and used to puncture the umbilical vein ofa placenta obtained from healthy delivery from a mother tested for viraland bacterial infections according to international donor standards.Cord blood is allowed to drain by gravity so as to drip into the bloodbag. The placenta is placed in a plastic-lined, absorbent cotton padsuspended from a specially constructed support frame in order to allowcollection and reduce the contamination with maternal blood and othersecretions, The 63 ml of CPD A used in the standard blood transfusionbag, calculated for 450 ml of blood, is reduced to 23 ml by draining 40ml into a graduated cylinder just prior to collection. This volume ofanticoagulant matches better the cord volumes usually retrieved (<170ml).

An aliquot of the blood is removed for safety testing according to thestandards of the National Marrow Donor Program (NMDP) guidelines. Safetytesting includes routine laboratory detection of human immunodeficiencyvirus 1 and 2, human T-cell lymphotropic virus I and II, Hepatitis Bvirus, Hepatitis C virus, Cytomegalovirus and Syphilis. Subsequently, 6%(wt/vol) hydroxyethyl starch is added to the anticoagulated cord bloodto a final concentration of 1.2%.

The leukocyte rich supernatant is then separated by centrifuging thecord blood hydroxyethyl starch mixture in the original collection bloodbag (50×g for 5 min at 10° C.). The leukocyte-rich supernatant isexpressed from the bag into a 150-ml Plasma Transfer bag (Baxter HealthCare) and centrifuged (400×g for 10 min) to sediment the cells. Surplussupernatant plasma is transferred into a second plasma Transfer bagwithout severing the connecting tube. Finally, the sedimented leukocytesare resuspended in supernatant plasma to a total volume of 20 ml.Approximately 5×10⁸-7×10⁹ nucleated cells are obtained per cord. Cellsare cryopreserved according to the method described by Rubinstein et al(Rubinstein, et al. Processing and cryopreservation ofplacental/umbilical cord blood for unrelated bone marrow reconstitution.Proc Natl Acad Sci U S A 92:10119-10122). for subsequent cellulartherapy. CD34 cells are expanded by culture. CD34+ cells are purifiedfrom the mononuclear cell fraction by immuno-magnetic separation usingthe Magnetic Activated Cell Sorting (MACS) CD34+ Progenitor CellIsolation Kit (Miltenyi-Biotec, Auburn, Calif.) according tomanufacturer's recommendations. The purity of the CD34+ cells obtainedranges between 95% and 98%, based on Flow Cytometry evaluation (FACScanflow cytometer, Becton-Dickinson, Immunofluorometry systems, MountainView, Calif.). Cells are plated at a concentration of 10.sup.4 cells/mlin a final volume of 0.5 ml in 24 well culture plates (Falcon; BectonDickinson Biosciences) in DMEM supplemented with the cytokine cocktailof: 20 ng/ml IL-3, 250 ng/ml IL-6, 10 ng/ml SCF, 250 ng/ml TPO and 100ng/ml flt-3L and a 50% mixture of LPCM. LPCM is generated by obtaining afresh human placenta from vaginal delivery and placing it in a sterileplastic container. The placenta is rinsed with an anticoagulant solutioncomprising phosphate buffered saline (Gibco-Invitrogen, Grand Island,N.Y.), containing a 1:1000 concentration of heparin (1% w/w) (AmericanPharmaceutical Partners, Schaumburg, Ill.). The placenta is then coveredwith a DMEM media (Gibco) in a sterile container such that the entiretyof the placenta is submerged in said media, and incubated at 37.degree.C. in a humidified 5% CO.sub.2 incubator for 24 hours. At the end of the24 hours, the live placenta conditioned medium (LPCM) is isolated fromthe container and sterile-filtered using a commercially availablesterile 0.2 micron filter (VWR). Cells are expanded, checked for purityusing CD34-specific flow cytometry and immunologically matched torecipients using a mixed lymphocyte reaction. Cells eliciting a lowlevel of allostimulatory activity to recipient lymphocytes are selectedfor transplantation. Cells are administered as described above.

Example 2

Patient 242 was diagnosed with dilated, non-ischemic cardiomyopathy in2002 with an ejection fraction of approximately 30% as measured byechocardiogram (ECHO). The clinical presentation at diagnosis wasindicative of congestive heart failure, including marked dyspnea,inability to exercise, dizziness, and irregular heart beat. New YorkHeart Association (NYHA) classification of II. ECHO analysis in April2003 indicated ejection fraction of approximately 40%. Quality of lifeassessment using the Minnesota Living with Heart Failure Questionnaire(Middel, Bouma et al. 2001) revealed a score of 90. The patient wastreated under informed consent in December 2006 with a combination ofcord blood expanded allogeneic CD34 cells (2.5 million) and placentallyderived allogeneic mesenchymal stem cells (3 million) 3 times over theperiod of a week. Ascorbic acid was administered intravenously duringthis period at a concentration of 25 grams intravenously given in 250 mlof saline over a 30 to 40 minute period on day 1 and 7 of treatment.

Cellular therapy was well tolerated and no adverse side effects wereobserved either acutely or as of this writing. The patient did notexperience either symptoms of either acute (skin rash of diarrhea) orchronic (skin rash, skin inflammation, mouth lesions, hair loss,indigestion) graft vs. host disease. Two weeks prior to anechocardiogram in April 2007, the patient voluntarily discontinued allabove-mentioned medications and supplements. The echocardiogram revealedan ejection fraction of 50-55%. The patient reports profound clinicalbenefit at time of writing (April , 2008), including resolution ofheart-failure associated symptoms of dizziness, fatigue, dyspnea, rapidheart beat, irregular heart beat, depression, blackouts, and loss ofsleep secondary to orthopnea. The Minnesota Living with Heart FailureQuestionnaire score was zero. The patient has a normal ejection fractionand no symptoms of heart failure and is no longer classifiable on theNYHA scale.

The invention may be embodied in other specific forms besides and beyondthose described herein. The foregoing embodiments are therefore to beconsidered in all respects illustrative rather than limiting, and thescope of the invention is defined and limited only by the appendedclaims and their equivalents, rather than by the foregoing description.

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What is claimed is:
 1. A method of amerliorating the effects ofmyocardial infarction comprising: a) identifying a subject who hassuffered from a myocardial infarction b) harvesting mesenchymal stemcells; c) intravenously injecting said mesenchymal stem cells into saidsubject; and d) administering ascorbic acid or a derivative thereofintravenously to said subject to ameliorate the effects of myocardialinfarction.
 2. The method of claim 1, wherein said ascorbic acid isadministered intravenously in an amount of 15-700 g/week.
 3. The methodof claim 2, wherein said ascorbic acid is administered intravenously inan amount of 25 g/day.
 4. The method of claim 1, wherein said ascorbicacid is intravenously administered to said subject prior to theadministration of said mesenchymal stem cells.
 5. The method of claim 1,further comprising measuring oxidative stress in said subject prior toadministration of said mesenchymal stem cells and wherein the ascorbicacid is intravenously administrated at a concentration and frequencybased upon said measurement of oxidative stress.
 6. The method of claim1, wherein the subject is tested after being administered the ascorbicacid and mesenchymal cells and there is an improvement in ejectionfraction and clinical heart failure score.
 7. The method of claim 1,wherein the subject is tested after being administered the ascorbic acidand mesenchymal stem cells and there is an improvement in ejectionfraction.
 8. The method of claim 1, wherein the subject is tested afterbeing administered the ascorbic acid and mesenchymal stem cells andthere is an improvement in clinical heart failure score.